Among closely related orbiviruses (Reoviridae family), such as Bluetongue virus (BTV), Epizootic haemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV), the latter is known to cause the most severe morbidity and mortality in infected animals. In susceptible horses, the AHSV can cause different forms of disease ranging from mild fever to an acute fever where mortality reaches 95-100%. Although AHSV is endemic to sub-Saharan Africa, occasional outbreaks have been reported in North Africa, Pakistan, India, Spain and Portugal, and these outbreaks have had significant social and economic impact. The afore three orbiviruses are transmitted by midges of the genus Culicoides that are also found throughout Europe and the USA, thus increasing the potential geographic risk of AHSV outbreak, as, indeed, has been reported for BTV and EHDV.
The genome sequence of AHSV is divergent from BTV and EHDV. Only nine serotypes of the AHSV virus, AHSV1-AHSV9, have been identified. Vaccination with a live-attenuated polyvalent AHSV vaccine (LAV) is currently used to control the disease in Africa. However, this vaccine is considered unsafe due to adverse side effects; it causes viremia and it also has the potential to re-assort with field isolates thus risking the introduction of new virus serotypes into naïve geographical locations.
Therefore, there is a need for a rationally designed safe AHSV vaccine. Challengingly, the likely obstacles in the development of a safe vaccine are fundamental to its characteristics, besides the need for safe and efficient vaccine preparation the vaccine must be: (i) technically simple and reproducible, this is crucial for upscaling of production; (ii) cost-effective, which is directly dependent on: the number of virus like particles (VLPs) produced per cell, the heterologous nature of the vaccines, as well as the easiness of downstream processing. Unfortunately, subunit vaccines based on purified proteins, generally do not provide sufficient and long-lasting protection, moreover, they are costly to produce.
AHSV is a non-enveloped virus containing 10 double-stranded RNA (dsRNA) segments (S1-S10). The outer capsid is composed of two major structural proteins VP2 and VP5. The serotype-determining VP2 is the most variable viral protein, being the major target of a protective immune response. The core particle consists of two concentric layers: the surface VP7 layer and the inner VP3 layer, which enclose the 10 dsRNAs and a complex of replication enzymes VP1, VP4 and VP6. Comparisons between the sequences of the capsid proteins VP2, VP3, VP5 and VP7 of BTV10 with those of EHDV1 and AHSV4 have revealed the close relationship between these viruses: the inner core proteins, VP3 and VP7, are the most conserved, whereas outermost proteins, VP2 and VP5, are the most variable. Whilst previous structural studies of BTV have revealed details of BTV particle organization, currently, only the structure of a (top) domain of VP7 has been reported for AHSV, therefore analysis of AHSV virus functioning and any subsequent rational structure-based vaccine design is not possible for AHSV.
In the past decade, reverse genetics (RG) technology has revolutionized the understanding of viral replication and pathogenesis, and made a great contribution towards the development of vaccine technologies. However, whilst the RG technology for wildtype AHSV strains has been developed, it needs significant improvement to increase the efficiency of rescued virus production.
Herein we disclose the creation of mutated, replication-competent, propagation-disabled virus particles. Notably, passaging in a complementary cell line did not reveal changes in the mutated segment, which would restore any mutated ORFs. Further, replication efficiencies were close to wild-type (wt) AHSV thus generating substantial protein levels for vaccine development. This therefore represents a stable, superior and efficient vaccine therapy.
As a proof of principle, vaccination of IFNAR−/− mice with said vaccine and challenged with two virulent strains of AHSV showed efficient protection against homologous infection. Moreover, vaccination of AHSV natural hosts, e.g. ponies with a monoserotype (ECRA.A4) vaccine and one multivalent cocktail (ECRA.A1/4/6/8) vaccine showed protection against a virulent AHSV4. Furthermore, the multivalent cocktail vaccinated ponies produced neutralizing antibodies against all the serotypes present in the cocktail, and a foal born during the trial was healthy and had no viremia. These results validate the suitability of the technology as vaccines for AHSV.